1. Field of the Invention
The present invention relates to the fields of retinoic acid receptor (RAR) biology and transgenic mice. Specifically, the present invention relates to mice which are deficient in the normal expression of one or more of the genes encoding members of the RAR or RXR class of receptors, to mice heterozygous for such deficiency, to cell lines, preferably pluripotent or totipotent cell lines, which are heterozygous or homozygous for such deficiency, and to methods of using said mice or said cell lines to identify agonists and antagonists of specific members of the RAR or RXR class of receptors.
2. Description of the Related Art
It has long been established that retinoids (vitamin A derivatives) are crucial for normal growth, vision, maintenance of numerous tissues, reproduction and overall survival (Wolbach, S. B., and Howe, P. R., J. Exp. Med. 42:753-777 (1925); for reviews and refs see Spom et al., The retinoids, Vols. 1 and 2, Sporn et al., eds., Academic Press, Orlando, Fla. (1984); Livrea and Packer, in Retinoids, Livrea and Packer, eds., Marcel Dekker, New York (1993)). In addition offspring of vitamin A deficient (VAD) dams exhibit a number of developmental defects, indicating that retinoids are also important during embryogenesis (Wilson, J. G., et al., Am. J. Anat. 92:189-217 (1953)). With the exceptions of vision (Wald, 1968) and possibly of spermatogenesis in mammals (Thompson et al., Proc. Royal Soc. 159:510-535 (1964); van Pelt, H. M. M., and De Rooij, D. G., Endocrinology 128:697-704 (1991); and refs therein), most of the effects generated by VAD in fetuses, young, and adult animals can be prevented and/or reversed by retinoic acid (RA) administration (Wilson, J. G., et al., Am. J. Anat. 92:189-217 (1953); Thompson et al., Proc. Royal Soc. 159:510-535 (1964)). The dramatic teratogenic effects of maternal RA administration on mammalian embryos (Shenefelt, R. E., Teratology 5, 103-108 (1972); Lammer et al., N. Eng. J. Med. 313:837-841 (1985); Webster, W. S. et al., J. Cranofac. Genet. Dev. Biol. 6:211-222 (1986); Kessel and Gruss, Cell 67:89-104 (1991); Kessel, M., Development 115:487-501 (1992); Creech Kraft, J., "Pharmacokinetics, placental transfer, and teratogenicity of 13-cis-retinoic acid, its isomer and metabolites," In Retinoids in Normal Development and Teratogenesis, G. M. Morriss-Kay, ed., Oxford University Press, pp. 267-280 (1992)), and the spectacular effects of topical administration of retinoids on embryonic development of vertebrates and limb regeneration in amphibians (Mohanty-Hejmadi et al., Nature 355:352-353 (1992); for review and refs see Tabin, C. J., Cell 66:199-217 (1991)), has markedly contributed to the belief that RA could in fact be a morphogen (conferring positional information during development), and may also play a critical role during organogenesis.
With the exception of visual perception (Wald, G. et al., Science 162:230-239 (1968)), the molecular mechanisms underlying the highly diverse effects of retinoids has remained obscure until recently. The discovery of nuclear receptors for RA (Petkovich et al., Nature 330:444-450 (1987); Giguere et al., Nature 330:624-629 (1987)) has greatly advanced the understanding of how these simple molecules could exert their pleiotropic effects (for reviews see Leid et al., TIBS 17:427-433 (1992); Linney, E., Current Topics in Dev. Biol. 27:309-350 (1992)). It is thought that the effects of the RA signal are mediated through two families of receptors which belong to the superfamily of ligand-inducible transcriptional regulatory factors that include steroid/thyroid hormone and vitamin D3 receptors (for reviews see Evans, R. M., Science 240:889-895 (1988); Green and Chambon, Trends Genet. 4:309-314 (1988); Beato, M., Cell 56:335-344 (1989); Gronemeyer, H., Ann. Rev. Genet. 25:89-123 (1991); de Luca, L. M., FASEB J. 5:2924-2933 (1991); Linney, E., Current Topics in Dev. Biol. 27:309-350 (1992); Yu, V. C. et al., Cur. Op. Biotech. 3:597-602 (1992); Leid et al., TIBS 17:427-433 (1992)).
The RAR family (RAR.alpha., .beta. and .gamma. and their isoforms) are activated by both all-trans and 9-cis RA, whereas the retinoid X receptor family (RXR.alpha., .beta. and .gamma.) are activated exclusively by 9-cis RA (for review and refs see de Luca, L. M., FASEB J. 5:2924-2933 (1991); Linney, E., Current Topics in Dev. Biol. 27:309-350 (1992); Yu, V. C. et al., Cur. Op. Biotech. 3:597-602 (1992); Leid et al., TIBS 17:427-433 (1992); Kastner et al., "The role of nuclear retinoic acid receptors in the regulation of gene expression," in Vitamin A in health and disease, R. Blomhoff, ed., Marcel Dekker, New York (1993); Allenby et al., Proc. Natl. Acad. Sci. USA 90:30-34 (1993)). Within a given species, the DNA binding (region C) and the ligand binding (region E) domains of the three RAR types are highly similar, whereas the C-terminal region F and the middle region D exhibit no or little similarity. The amino acid sequences of the three RAR types are also notably different in their B regions, and their main isoforms (.alpha.1 and .alpha.2, .beta.1 to .beta.4, and .gamma.1 and .gamma.2) further differ in their N-terminal A regions (reviewed in Leid et al., TIBS 17:427-433 (1992)). Similarly, the RXRs characterized to date also markedly differ in their N-terminal A/B regions (Leid et al., TIBS 17:427-433 (1992); Leid et al., Cell 68:377-395 (1992); Mangelsdorf et al., Genes and Dev. 6:329-344 (1992)). Amino acid sequence comparisons revealed that the interspecies conservation of a given RAR or RXR type is greater than the similarity found between the three RAR or RXR types within a given species (reviewed in Leid et al., TIBS 17:427-433 (1992)). This interspecies conservation is particularly striking in the N-terminal A regions of the various RAR.alpha., .beta. and .gamma. isoforms, whose A region amino acid sequences are very divergent from each other. Taken together with the distinct spatio-temporal expression patterns observed for the transcripts of each RAR and RXR type in the developing embryo and various adult mouse tissues (Zelent, A., et al., Nature 339:714-717 (1989); Dolle et al., Nature 342:702-705 (1989); Dolle et al., Development 110:1133-1151 (1990); Ruberte et al., Development 108:213-222 (1990); Ruberte et al., Development 111:45-60 (1991); Mangelsdorf et al., Genes and Dev. 6:329-344 (1992)) this interspecies conservation has suggested that each RAR and RXR type (and isoform) may perform unique functions. This hypothesis is further supported by the finding that the various RAR isoforms and RXR types contain two transcriptional activation functions (AFs) located in the N-terminal A/B region (AF-1) and in the C-terminal E region (AF-2), which can synergistically, and to some extent differentially, activate various RA-responsive promoters (Leid et al., TIBS 17:427-433 (1992); Nagpal et al., Cell 70:1007-1019 (1992); Nagpal et al., EMBO J., in press (1993)). Moreover, it has been shown that activation of RA-responsive promoters likely occurs through RAR:RXR heterodimers rather than through homodimers (Yu, V. C. et al., Cell 67:1251-1266 (1991); Leid et al., Cell 68:377-395 (1992b); Durand et al., Cell 71:73-85 (1992);Nagpal et al., Cell 70:1007-1019 (1992); Zhang, X. K., et al., Nature 355, 441-446 (1992); Kliewer et al., Nature 355:446-449 (1992); Bugge et al., EMBO J. 11:1409-1418 (1992); Marks et al., EMBO J. 11:1419-1435 (1992); for reviews see Yu, V. C. et al., Cur. Op. Biotech. 3:597-602 (1992); Leid et al., TIBS 17:427-433 (1992); Laudet and Stehelin, Curr. Biol. 2:293-295 (1992); Green, S., Nature 361:590-591 (1993)). Thus, the basis for the highly pleiotropic effect of retinoids may reside, at least in part, through the control of different subsets of retinoid-responsive promoters by cell-specifically expressed heterodimeric combinations of RAR:RXR types (and isoforms), whose activity may be regulated by cell-specific levels of all-trans and 9-cis RA (Leid et al., TIBS 17:427-433 (1992).
The apparently ubiquitous distribution of RAR.alpha. transcripts (mainly the RAR.alpha.1 isoform; Zelent, A., et al., Nature 339:714-717 (1989); Leroy et al., EMBO J. 10:59-69 (1991); Leroy et al., Proc. Natl. Acad. Sci. USA 88:10138-10142 (1991); Dolle et al., Nature 342:702-705 (1989); Dolle et al., Development 110:1133-1151 (1990); Ruberte et al., Development 111:45-60 (1991); E. Ruberte, P. Dolle, D. Decimo and P. C., unpublished results) during development and in adult tissues suggests that RAR.alpha.1 may play some general housekeeping function (Brand et al., Nucl. Acid Res. 18:6799-6806 (1990); Leroy et al., EMBO J. 10:59-69 (1991)). RAR.beta. transcripts exhibit a more restricted pattern of distribution in developing embryos and adult tissues, suggesting that RAR.alpha. isoforms could be involved in the differentiation of certain epithelia, as well as in the ontogenesis of the nervous system (Dolle et al., Development 110:1133-1151 (1990); Ruberte et al., Development 111:45-60 (1991); Mendelsohn et al., Development 113:723-734 (1991)). In situ hybridization studies indicate that RAR.gamma. transcripts are apparently restricted to the presomitic caudal region of day 8.0 p.c. embryos, and to the frontonasal mesenchyme, pharyngeal arches, sclerotomies and limb bud mesenchyme at day 8.5 to 11.5 p.c. At later stages, RAR.gamma. transcripts are found in precartilaginous condensations (day 12.5 p.c.), with subsequent restriction to cartilage and differentiating squamous keratinizing epithelia (day 13.5 p.c.), regardless of their embryonic origin (Dolle et al., Nature 342:702-705 (1989); Dolle et al., Development 110:1133-1151 (1990); Ruberte et al., Development 108:213-222 (1990)). These observations suggest a role for RAR.gamma. in morphogenesis, chondrogenesis and differentiation of squamous epithelia (Dolle et al., Development 110:1133-1151 (1990); Ruberte et al., Development 108:213-222 (1990)). In addition, Northern blot analysis indicates that the RAR.gamma.2 isoform is the predominant isoform in the early embryo (day 8.5 to 9.5 p.c.), whereas RAR.gamma.1 is the predominant RAR.gamma. isoform transcript found later in embryogenesis as well as in newborn and adult skin (Kastner et al., Proc. Natl. Acad. Sci. USA 87:2700-2704 (1990)).
The mouse is the model of preference in the study of the mammalian genetic system, and a great deal of research has been performed to map the murine genome.
It would be of great importance to be able to establish a living model wherein the role of the various members of the RAR class of receptors could be studied in a definitive manner.
Accordingly, it is an object of the present invention to generate strains of mice which do not express, or express at undetectable levels, one or more members of the RAR or RXR class of receptors. Such mice would be of great value for a better understanding of the role each of the members of the RAR or RXR class of receptors because such animals and cell lines would allow direct testing of the function of specific genes, either deleted or reintroduced by transgenesis, and would serve as an assay system to identify compounds which act as antagonists or agonists of specific members of the RAR or RXR class of receptors.